Neurotrophic Peptides

ABSTRACT

The present invention relates to a neurotrophic peptide having an amino acid sequence selected from the group consisting of VG-DGGLFEKKL (SEQ ID No. 1), EDQQVHFTPTEG (SEQ ID No. 2) and IPENEADGMPATV (SEQ ID NO. 3).

The present invention relates to neurotrophic and/or neurogenic peptidesand their use for manufacturing a medicament for the treatment ofneurodegenerative diseases.

The population in the industrialised countries is rapidly ageing due toa greater life expectancy, and an ever-increasing number of people areafflicted with neurodegenerative diseases making a global issue out ofthese diseases.

Neurodegenerative diseases result from the gradual and progressive lossof neural cells, leading to nervous system dysfunction, and may havenext to ageing various causes (e.g. environmental influences, geneticdefects). Till now more than 600 neurologic disorders are known.

The major known risk factors for neurodegenerative disease includecertain genetic polymorphisms and increasing age. Other possible causesmay include gender, poor education, endocrine conditions, oxidativestress, inflammation, stroke, hypertension, diabetes, smoking, headtrauma, depression, infection, tumors, vitamin deficiencies, immune andmetabolic conditions, and chemical exposure. Because the pathogenesis ofmany of these diseases remains unknown, also the role of environmentalfactors in these diseases may be considered. An overview ofneurodegenerative diseases can be found, for instance, in“Neurodegenerative Diseases: Neurobiology, Pathogenesis andTherapeutics” (M. Flint Beal, Anthony E. Lang, and Albert C. Ludolph;Cambridge University Press; 2005).

In order to treat neurodegenerative diseases several medicamentscomprising one or more active compounds like Piracetam, Nimotop,Vinpocetin, Gliatilin, Cerebrolysin, Cytoflavin etc. are regularlyemployed. The compounds known in the art have varying modes of action.Cerebrolysin, for instance, a peptide based drug produced from purifiedanimal brain proteins by standardized enzymatic breakdown, is exertingnerve growth factor like activity on neurons from dorsal root ganglia,neurotrophic and neuroprotective effects.

US 2004/102370 relates to peptides comprising the essential tetramericpeptide structural unit Xaa-Xaa-Xaa-Xaa in which Xaa at position 1represents Glu or Asp, Xaa at position 2 represents any amino acid, Xaaat position 3 represents any amino acid and Xaa at position 4 representsGlu or Asp. Said peptides are used to treat neurodegenerative diseasesand nerve damages, and are described to be stimulators of axonalregeneration and survival.

Ciliary neurotrophic factor (CNTF) is a survival factor for variousneuronal cell types. The human CNTF protein comprises 200 amino acidresidues and shares significant sequence homology with CNTF proteinsfrom other mammalian sources. The gene for human CNTF has been clonedand recombinant forms of the protein are available for clinical trialsin humans (WO 91/04316). Over the past decade, a number of biologicaleffects have been ascribed to CNTF in addition to its ability to supportthe survival of ciliary ganglion neurons. CNTF is believed to induce thedifferentiation of bipotential glial progenitor cells in the perinatalrat optic nerve and brain (Hughes et al., 1988, Nature 335:70-73).Furthermore, it has been observed to promote the survival of embryonicchick dorsal root ganglion sensory neurons (Skaper and Varon, 1986,Brain Res. 389:39-46). In addition, CNTF supports the survival anddifferentiation of motor neurons, hippocampal neurons and presympatheticspinal cord neurons (Sendtner, et al., 1990, Nature 345: 440-441).

In addition to human CNTF, the corresponding rat and rabbit genes havebeen cloned and found to encode a protein of 200 amino acids, whichshare about 80% sequence identity with the human gene.

Despite their structural and functional similarity, recombinant humanand rat CNTF differ in several respects. The biological activity ofrecombinant rat CNTF in supporting survival and neurite outgrowth fromembryonic chick ciliary neurons in culture is four times better thanthat of recombinant human CNTF (Masiakowski et al., 1991, J. Neurochem.57:1003-1012). Further, rat CNTF has a higher affinity for the humanCNTF receptor than does human CNTF.

As described in WO 99/43813 one of the uses of CNTF is the use of CNTFfor the treatment of Huntington's disease. Huntington's disease (HD) isan hereditary degenerative disorder of the central nervous system.

However, the administration of CNTF to the human body has severaldrawbacks. While its therapeutic potential for CNS diseases is wellrecognized, the blood brain barrier (BBB) hinders the systemic deliveryof CNTF and direct bolus injections are not suitable due to the shorthalf-life of CNTF. One method of overcoming the blood brain barrierwhile providing continuous delivery of CNTF is, e.g., withimmunoisolated cellular implants that produce and deliver CNTF directlyto the region of interest. Cells can be protected from host rejection byencapsulating, or surrounding, them within an immunoisolatory,semipermeable membrane that admits oxygen and required nutrients andreleases bioactive cell secretions, but restricts passage of largercytotoxic agents from the host immune defense system. The selectivemembrane eliminates the need for chronic immunosuppression of the hostand allows the implanted cells to be obtained from nonhuman sources.However, also this method is not advantageous.

It is an object of the present invention to provide new medicamentscomprising substances which have substantially the same or even betterneurotrophic and/or neurogenic effects than CNTF. Advantageously thesesubstances should also be able to pass the blood brain barrier in orderto reach the wanted site of action in the brain.

Therefore, the present invention relates to a neurotrophic and/orneurogenic peptide having an amino acid sequence selected from the groupconsisting of VGDGGLFEKKL (SEQ ID No. 1), EDQQVHFTPTEG (SEQ ID No. 2) orIPENEADGMPATV (SEQ ID No. 3).

It has surprisingly been found that the peptides of the presentinvention, which are derivable from rat or human CNTF, show neurotrophicand/or neurogenic (causing growth of nerve tissue) effects which arecomparable to the wild-type CNTF. Furthermore due to their small sizethese peptides are also able to pass the blood brain barrier.

Fragments of SEQ ID No. 1 to 3 preferably comprise 4 to 10, morepreferably 4 to 8, even more preferably 4 to 6, amino acids and include:

Source Sequence SEQ ID No. SEQ ID No. 1 GDGGLFEK 5 GLFEKKLW 6 VGDG 7GDGG 8 DGGL 9 GGLF 10

The peptides of the present invention and their fragments may be fusedto other proteins, polypeptides or peptides (N- or C-terminally), orconjugated to other substances. The resulting fusions may also comprisemore than one peptide of the present invention (e.g. SEQ ID No. 1 may befused to SEQ ID No. 2). The peptides of these polypeptides may be fuseddirectly or via a linker to each other. Therefore, the present inventionalso relates to a polypeptide comprising at least two, preferably atleast three, peptides of the present invention (SEQ ID No. 1 to 10).

The peptides of the present invention may also be bound or conjugated tosubstances which enhance their ability to pass through the blood brainbarrier.

“Fragments”, as used herein, refer to parts of the peptides of thepresent invention, which are directly derivable from said peptides andshow the same as or enhanced neurotrophic and neurogenic activities thanthe wild-type CNTF.

According to the present invention also peptides are encompassed whichexhibit at least 80%, preferably 90%, more preferably 95%, identity withthe peptides of the present invention selected from the group consistingof SEQ ID No. 1 to 3.

According to the present invention “identity” (“identical”) isdetermined by comparing two optimally aligned sequences over acomparison window, where the fragment of the amino acid sequence in thecomparison window may comprise additions or deletions (e.g., gaps oroverhangs) as compared to the reference sequence (which does notcomprise additions or deletions) for optimal alignment of the twosequences. In general, sequences are aligned so that the highest ordermatch is obtained (see, e.g.: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991; Carillo et al. (1988) SIAM J AppliedMath 48:1073).

Whether any two amino acid molecules have amino sequences that are atleast, for example, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%“identical”, can be determined using known computer algorithms such asthe “FAST A” program, using for example, the default parameters as inPearson et al. (1988) PNAS USA 85: 2444 (other programs include the GCGprogram package (Devereux, J., et al., Nucleic Acids Research (1984)Nucleic Acids Res., 12, 387-395), BLASTP, BLASTN, FASTA (Atschul, S. F.,et al., J Molec Biol 215: 403 (1990); Guide to Huge Computers, Martin J.Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al, (1988)SIAM J Applied Math 48: 1073). For instance, the BLAST tool of the NCBIdatabase can be used to determine identity. Other commercially orpublicly available programs include, DNAStar “MegAlign” program(Madison, Wis.) and the University of Wisconsin Genetics Computer Group(UWG) “Gap” program (Madison Wu)). Percent identity of proteins and/orpeptides can be determined, for example, by comparing sequenceinformation using a GAP computer program (e.g. Needleman et al., (1970)J. Mol. Biol. 48:443, as revised by Smith and Waterman (1981) Adv. Appl.Math. 2:482). Briefly, the GAP program defines similarity as the numberof aligned symbols (i.e., nucleotides or amino acids) which are similar,divided by the total number of symbols in the shorter of the twosequences. Default parameters for the GAP program can include: (1) aunary comparison matrix (containing a value of 1 for identities and fornon-identities) and the weighted comparison matrix of Gribskov et al.14:6745, as described by Schwartz and Dayhoff, eds., ATLAS OF PROTEINSEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp.353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10penalty for each symbol in each gap; and (3) no penalty for end gaps.

As used herein, the term “at least 80% identical to” refers to percentidentities from 80 to 99.99 relative to the reference peptides.Consequently, the peptides of the present invention may also compriseone or more amino acid modifications (i.e. substitutions, deletions,insertions) provided that the peptides still exhibit neurotrophic and/orneurogenic activity.

Identity at a level of 80% or more is indicative of the fact that,assuming for exemplification purposes a test and reference polypeptidelength of 100 amino acids is compared, no more than 20% (i.e. 20 out of100) of amino acid residues in the test polypeptide differs from that ofthe reference polypeptide. Such differences can be represented as pointmutations randomly distributed over the entire length of an amino acidsequence or they can be clustered in one or more locations of varyinglength up to the maximum allowable, e.g. 20/100 amino acid difference(approx. 80% identity). Differences are defined as amino acidsubstitutions, insertions or deletions. At the level of homologies oridentities above about 85-90%, the result should be independent of theprogram and gap parameters set; such high levels of identity can beassessed readily, often without relying on software.

According to a preferred embodiment of the present invention theneurotrophic peptide of the present invention is identical to SEQ ID No.1, 2 or 3, which means that the neurotrophic peptide consists of saidamino acids sequences or fragments thereof. Of course, the peptide ofthe present invention may comprise modifications such as substitution ofL-amino acids with D-amino acids, introduction of hydrophobic sidechains, modifications allowing the formation of dimers (or evenmultimers) or cyclic peptide variants. The respective methods are wellknown in the art.

The peptide according to the present invention is preferably nonimmunogenic. The term “non immunogenic peptide” as used herein refers toa molecule, in particular to a peptide, which does substantially notprovoke an immune response in vivo when administered to a human or ananimal being. This molecule property can be determined by methods knownin the art. For instance, if the administration of a molecule accordingto the present invention to an animal (e.g. rabbit, mouse) provokes inan animal a substantial increase of antibodies directed against saidmolecule, said molecule is considered as an “immunogenic peptide”, if,however, substantially no molecule-specific anti-bodies can be inducedin an animal or human upon administration of said molecule, it isconsidered as a “non immunogenic peptide”. It is important that thepeptides according to the present invention are non immunogenic becauseimmunogenic peptides are normally eliminated from the body by the immunesystem.

The basic structure of the peptide according to the present invention,which is formed by amino acids, is preferably synthesised chemicallyaccording to methods known in the art, e.g. by the method developed byMerrifield et al. (Merrifield, R. B. (1963) J. Am. Chem. Soc. 85,2149-2154; solid phase peptide synthesis).

The solid phase peptide synthesis method introduced by Merrifield in1963, for instance, involves the attachment of a growing peptide chainto a solid support. An amino acid corresponding to the C-terminal of thetarget peptide is covalently attached to an insoluble polymeric support(the “resin”). The next amino acid, with a protected alpha-amino acid,is activated and reacted with the resin-bound amino acid to yield anamino-protected dipeptide on the resin. The amino-protecting group isremoved and chain extension is continued with the third and subsequentprotected amino acids. After the target protected peptide chain has beenbuilt up the resin is cleaved by suitable chemical means therebyreleasing the crude peptide product into solution (for solid phasepeptide synthesis methods and other peptide synthesis methods see alsoFields, G. B. (ed.), Solid-Phase Peptide Synthesis in Methods inENZYMOLOGY, Vol. 289, Academic Press, San Diego (1997); Bodansky, M.,Bodansky, A., The practice of peptide synthesis (2nd edn.), SpringerVerlag, Berlin (1995); Pennington, M. W., Dunn, B. M. (eds), PeptideSynthesis Protocols, in Methods in Molecular Biology, Vol. 35, HumanaPress Inc., Totowa (1994); Grant, G. A. (ed.), Synthetic peptides: auser's guide, W.H. Freemann & Co., New York (1992)).

The inorganic cation at the C-terminal end of the peptide according tothe present invention may be an alkali metal or alkali earth metalcation, preferably a lithium, sodium, potassium, magnesium or calciumcation.

These inorganic cations are regularly used to prepare salts ofpharmaceutically active substances.

The organic cation may be a quaternary ammonium ion.

If the N-terminal end of the peptide according to the present inventioncomprises a positive charge, said charge may be preferably compensatedby an equivalent of an inorganic or organic anion. The organic anion canbe, for instance, acetate anion.

Of course it is also possible to use molecules, preferably smallmolecules, mimicking the peptides of the present invention.

Another aspect of the present invention relates to a pharmaceuticalcomposition comprising at least one peptide according to the presentinvention and/or at least one peptide having an amino acid sequenceselected from the group consisting of GDGGLFEK (SEQ ID No. 5), GLFEKKLW(SEQ ID No. 6), VGDG (SEQ ID No. 7), GDGG (SEQ ID No. 8), DGGL (SEQ IDNo. 9) and GGLF (SEQ ID No. 10) and optionally at least onepharmaceutically acceptable excipient and/or carrier.

The peptide according to the present invention may be formulated in apharmaceutical preparation, which can be administered to a patient forpreventing or treating a cerebral disease, in particular aneurodegenerative disease. The pharmaceutical preparation may furthercomprise pharmaceutically acceptable excipients and/or carriers.Suitable excipients and carriers are well known in the art (see e.g.“Handbook of Pharmaceutical Excipients”, 5th Edition by Raymond C. Rowe,Paul J. Sheskey, Siân C. Owen (2005), APhA Publications).

The composition of the present invention may further comprise at leastone additional pharmaceutically active component, which is preferablyIPRNEADGMPINV (SEQ ID No. 4).

The pharmaceutical preparation according to the present invention maycomprise next to the peptide according to the present invention furtheractive components, which may exhibit similar properties whenadministered to an individual or which may cause other reactions in thetreated patient.

According to the present invention, e.g., antioxidants like vitamins maybe considered as further active components because antioxidants inhibitoxidation or suppress reactions promoted by oxygen, oxygen freeradicals, oxygen reactive species including peroxides. Antioxidants,especially lipid-soluble antioxidants, can be absorbed into the cellmembrane to neutralize oxygen radicals and thereby protect the membrane.The antioxidants useful in the present invention are preferably vitaminantioxidants that may be selected from the group consisting of all formsof Vitamin A including retinal and 3,4-didehydroretinal, all forms ofcarotene such as alpha-carotene, beta-carotene, gamma-carotene,delta-carotene, all forms of Vitamin C (D-ascorbic acid, L-ascorbicacid), all forms of tocopherol such as Vitamin E (Alpha-tocopherol,3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltri-decyl)-2H-1-benzopyran-6-ol),beta-tocopherol, gamma-tocopherol, delta-tocopherol, tocoquinone,tocotrienol and Vitamin E esters which readily undergo hydrolysis toVitamin E such as Vitamin E acetate and Vitamin E succinate, andpharmaceutically acceptable Vitamin E salts such as Vitamin E phosphate,prodrugs of Vitamin A, carotene, Vitamin C, and Vitamin E,pharmaceutically acceptable salts of Vitamin A, carotene, Vitamin C, andVitamin E, and the like, and mixtures thereof.

According to another preferred embodiment of the present invention thecomposition is provided for intravenous, intramuscular, spinal,epidural, transdermal, intranasal, mucosal, parenteral, oral, enteral orrectal administration.

Depending on the route of administration the pharmaceutical compositionaccording to the present invention may be formulated, for instance, astablets, capsules, liquids, infusion and suppositories (see e.g.“Pharmaceutical Formulation Development of Compounds” by Sven Frokjaer(1999), CRC; “Handbook of Pharmaceutical Manufacturing Formulations” bySarfaraz K. Niazi (2004), CRC).

The peptides are preferably comprised in the composition in an amountbetween 0.1 μg/g to 100 mg/g, preferably 1 μg/g to 80 mg/g. In any way,the effective dosages for prevention or treatment of human patients canbe optimised for given patients or patient collectives according to theroutine methods available for the present field.

Another aspect of the present invention relates to the use of at leastone peptide with neurotrophic and/or neurogenic activity as definedabove which may be part of a molecule consisting of a maximum of 50,preferably a maximum of 40, more preferred a maximum of 30, even morepreferred a maximum of 20, amino acids, and/or at least one peptidehaving an amino acid sequence selected from the group consisting ofG-D-G-G-L-F-E-K (SEQ ID No. 5), G-L-F-E-K-K-L-W (SEQ ID No. 6), V-G-D-G(SEQ ID No. 7), G-D-G-G (SEQ ID No. 8), D-G-G-L (SEQ ID No. 9) andG-G-L-F (SEQ ID No. 10) for the manufacture of a medicament for thetreatment and/or prevention of a neurodegenerative disease.

According to the present invention all peptides disclosed herein andexhibiting neurotrophic and/or neurogenic activity may be used formanufacturing a medicament for the treatment and/or prevention ofneurodegenerative diseases.

According to a preferred embodiment of the present invention the peptideis a peptide according to the present invention as defined above.

The neurodegenerative disease is preferably selected from the groupconsisting of Alexander disease, Alper's disease, Alzheimer disease,Amyotrophic lateral sclerosis, Ataxia telangiectasia, Canavan disease,Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease,epilepsy, Huntington disease, Kennedy's disease, Krabbe disease, Lewybody dementia, Machado-Joseph disease (Spinocerebellar ataxia type 3),Multiple sclerosis, Multiple System Atrophy, Parkinson disease,Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral sclerosis,Refsum's disease, Sandhoff disease, Schilder's disease, Spinocerebellarataxia, Steele-Richardson-Olszewski disease, stroke, depression andTabes dorsalis.

Next to these preferred neurodegenerative diseases the peptide accordingto the present invention may also be used to treat other cerebraldisorders.

In one embodiment of the invention a peptide or protein comprising orconsisting of a peptide of the present invention can be employed as adrug stimulating cerebral reparative process and used for the treatmentand prevention of trauma-associated cerebral lesions, including thetreatment of cerebral lesions after a fracture of the cranial vault,skull base, multiple bone fractures, the treatment for the cerebrallesions in cases of intracranial trauma (e.g. posttraumatic cerebralconcussion, cerebral wounds and contusion, subarachnoid, subdural andextradural haemorrhage), the treatment and prevention of traumaticshock, the treatment of the cerebral lesions associated with the impactof radiation, lowered temperature, heat and light, air pressure,electric and ultrahigh frequency current, the treatment and preventionof delayed-onset effects of skull fractures, the treatment andprevention of delayed-onset effects of intracranial trauma, thetreatment and prevention of delayed-onset cerebral lesions induced byradiation, complications after surgical and other medical interventions.

In another embodiment of the present invention the peptides according tothe present invention may be used as a drug suppressing toxic effects ofthe neurotrophic agents, stimulating cerebral repair processes andrevealing cerebroprotective activity for the treatment and prevention ofcerebral lesions after poisoning including the treatment of cerebrallesions after poisoning with therapeutic agents, medicinal andbiological compounds, the treatment of the cerebral impairment withagents of non-medical origin, the treatment and prevention ofdelayed-onset cerebral lesions induced by poisoning with drugs andnon-medical substances.

In another embodiment of the present invention the peptides according tothe present invention may be used as drug with nootropic activity andstimulating cerebral repair processes for the treatment and preventionof mental deficiencies.

In another embodiment of the present invention the peptides according tothe present invention may be used for stimulating cerebral repairprocesses and motional activity for the treatment and prevention ofparalytic disorders including the treatment and prevention ofhemiplegia, the treatment and prevention of infantile cerebralparalysis, the treatment and prevention of other paralytic syndromes(quadriplegia, paraplegia, diplegia of upper extremities, monoplegia oflower extremities).

In another embodiment of the present invention the peptides according tothe present invention may be used as drug stimulating cerebral repairprocesses with cerebroprotective activity for the treatment andprevention of cerebral impairments in case of chromosome anomaliesincluding Down's syndrome.

In another embodiment of the present invention the peptides according tothe present invention may be used as drug stimulating cerebral repairprocesses with cerebroprotective activity for the treatment andprevention of cerebral impairments in case of inflammatory cerebraldisorders including the treatment and prevention of cerebral impairmentsin case of bacterial meningitis including cryptococcus meningitis inAIDS patients, the treatment and prevention of cerebral impairments incase of non-bacterial meningitis, the treatment and prevention ofcerebral impairments in case of meningitis of unclear origin, thetreatment and prevention of cerebral impairments in case ofencephalitis, myelitis and encephalomyelitis, including cerebraltoxoplasmosis in AIDS patients, for the treatment and prevention ofcerebral impairments in case of intracranial abscesses, for thetreatment and prevention of cerebral impairments in case of phlebitisand thrombophlebitis of intracranial venous sinus, for the treatment andprevention of sequalae after intracranial abscesses or purulentinfection.

In another embodiment of the present invention the peptides according tothe present invention may be used as drug stimulating cerebral repairprocesses with cerebroprotective and nootropic activity for thetreatment and prevention of cerebral impairments in case ofcerebral-vascular disorders including the treatment and prevention ofcerebral impairments in case of subarachnoid haemorrhage, treatment andprevention of cerebral impairments in case of cerebral haemorrhage, thetreatment and prevention of cerebral impairments in case of occlusionand Stenosis of precerebral arteries, the treatment and prevention ofcerebral impairments in case of occlusion of cerebral arteries, thetreatment and prevention of cerebral impairments in case of transitorycerebral ischemia, the treatment and prevention of cerebral impairmentsin case of other cerebral-vascular disorders (acute cerebral-vasculardisorders, cerebral atherosclerosis and other generalisedcerebral-vascular disorders, hypertension encephalopathy, cerebralaneurysm, cerebral arteritis and non-purulent thrombosis of intracranialvenous sinus).

In another embodiment of the present invention the peptides according tothe present invention may be used as drug stimulating cerebral repairprocesses, having cerebroprotective and nootropic activity for thetreatment and prevention of alcoholic psychosis including the treatmentand prevention of delirium tremens at abstinence syndrome, the treatmentand prevention of alcoholic amnestic syndrome and other alcoholicdementia disorders, the treatment and prevention of pathologic alcoholicintoxication, the treatment and prevention of alcoholic paranoia andalcoholic psychosis of paranoid type.

In another embodiment of the present invention the peptides according tothe present invention may be used as drug stimulating cerebral repairprocesses, having cerebroprotective and nootropic activity for thetreatment and prevention of cerebral impairment in case of alcoholism.

In another embodiment of the present invention the peptides according tothe present invention may be used as a drug suppressing toxic effects ofneurotropic agents and having cerebroprotective and nootropic activityfor the treatment and prevention of drug-induced psychosis including thetreatment and prevention of the drug abstinence syndrome, the treatmentand prevention of drug-induced paranoid and/or hallucinatory disorders,the treatment and prevention of pathologic intoxication with medicalagents, the treatment and prevention of other drug-induced psychicdisorders (delirium, dementia, amnestic syndrome and organic affectivesyndrome).

In another embodiment of the present invention the peptides according tothe present invention may be used as a drug suppressing toxic effects ofneurotropic agents and having cerebroprotective activity for thetreatment and prevention of drug addiction including the treatment andprevention of addiction to opioid agents, the treatment and preventionof addiction to barbiturate, sedative agents and tranquillisers, thetreatment and prevention of cocaine addiction, the treatment andprevention of addiction to cannabis and derivatives thereof, thetreatment and prevention of addiction to amphetamine andpsychostimulating agents, the treatment and prevention of addiction tohallucinogenic agents, treatment and prevention of cerebral impairmentscaused by drug abuse without drug addiction (abuse of alcohol, tobacco,cannabis, hallucinogens, opioids, cocaine, psychostimulating agents,antidepressants).

In another embodiment of the present invention the peptides according tothe present invention may be used as an agent for treatment andprevention of psychogenic symptoms and syndromes including the treatmentand prevention of psychogenic physiologic impairments, the treatment andprevention of other psychogenic symptoms and syndromes (stammering andimpediments, psychogenic anorexia tics, repeated stereotype movements,inorganic sleep disorders, psychogenic diet disorders, enuresis,psychalgia), the treatment and prevention of acute stress response, thetreatment and prevention of reactions induced by psychologicaldirections.

In another embodiment of the present invention the peptides according tothe present invention may be used as an agent for treatment andprevention of inorganic psychoses including the treatment and preventionof Schizophrenie disorders, the treatment and prevention of affectivepsychoses, the treatment and prevention of paranoid conditions, thetreatment and prevention of other inorganic psychoses (psychoses ofdepressive and agitate types, reactive confusion, acute paranoidreactions, psychogenic paranoid psychoses) and non-differentiatedpsychoses including psychoses induced with cerebral impairments in AIDSpatients, the treatment and prevention of infantile psychoses includinginfantile autism and disintegrative psychoses.

In another embodiment of the present invention the peptides according tothe present invention may be used as a drug stimulating cerebral repairprocesses and having cerebroprotective and nootropic activity for thetreatment and prevention of cerebral impairments in case of othercerebral disorders including the treatment and prevention of cerebralimpairments in case of cerebral cysts, the treatment and prevention ofhypoxic cerebral damage, the treatment and prevention of cerebralimpairments in case of intracranial hypertension, the treatment andprevention of cerebral impairments in case of encephalopathy.

In another embodiment of the present invention the peptides according tothe present invention may be used as drug stimulating cerebral repairprocesses and motional activity, having cerebroprotective and nootropiceffects for treatment and prevention of symptoms and syndromes in caseof various cerebral disorders including the treatment and prevention ofcognitive disorders, memory and artention, impairments (for instance, incase of amnestic diseases, mental deficiency, inorganic psychoses,etc.), the treatment and prevention of aphasia and apraxia (forinstance, in case of amnestic diseases, inorganic psychoses, cerebralimpairments due to chromosome anomalies, etc.), the treatment andprevention of emotional disorders (for instance, in case of inorganicpsychoses, demyelinising cerebral disorders, etc.), the treatment andprevention of psychopathologic syndrome (for instance, in case oftransitional organic psychotic conditions, drug-induced psychoses, drugaddiction, etc.), the treatment and prevention of asthenic-depressivesyndrome (for instance, in case of inorganic psychoses, cerebralimpairments due to chromosome anomalies, etc.), the treatment andprevention of delirium syndrome (for instance, in case of drug-inducedpsychoses and drug addiction, inorganic psychoses, etc.), the treatmentand prevention of sleep disorders (for instance, in case of cerebraltumours, transitional organic psychotic conditions, etc.), for treatmentand prevention of cerebral-focal syndrome (focal pathologic symptoms)(for instance, in case of cerebral impairments caused by complicationsof surgical or other medical intervention, demyelinising cerebraldisorders, etc.), the treatment and prevention of syndrome of motordisorders (for instance, in case of cerebral tumours, cerebralimpairments caused by poisoning, etc.), the treatment and prevention ofperipheral neuropathy, preferably diabetic neuropathy.

According to a preferred embodiment of the present invention themedicament further comprises a pharmaceutical acceptable excipientand/or carrier as defined above.

According to another preferred embodiment of the present invention thecomposition further comprises at least one additional pharmaceuticallyactive component.

The medicament is preferably provided for intravenous, intramuscular,spinal, epidural, transdermal, subcutaneous, intranasal, mucosal,parenteral, oral, enteral or rectal administration.

According to a preferred embodiment of the present invention themedicament comprises the peptide in an amount between 0.1 μg/g to 100mg/g, preferably 1 μg/g to 80 mg/g.

It is in particular preferred to use as peptide in a medicament of thepresent invention a peptide having the amino acid sequence SEQ ID No. 1,SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6,SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 and/or SEQ ID No. 10.

Another aspect of the present invention relates to a method forpreventing a break out of a neurodegenerative disease in an individualand for treating an individual suffering from a neurodegenerativedisease comprising the administration of a pharmaceutical composition orof an effective amount of at least one peptide according to the presentinvention.

The term “effective amount” of a peptide as used herein will dependamong other factors on the route of administration and physicalcondition of the individual to be exposed to said peptide. Methods forthe determination of the effective amount are known to the skilledperson.

The neurodegenerative disease is preferably selected from the groupconsisting of Alexander disease, Alper's disease, Alzheimer disease,Amyotrophic lateral sclerosis, Ataxia telangiectasia, Canavan disease,Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease,epilepsy, Huntington disease, Kennedy's disease, Krabbe disease, Lewybody dementia, Machado-Joseph disease (Spinocerebellar ataxia type 3),Multiple sclerosis, Multiple System Atrophy, Parkinson disease,Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral sclerosis,Refsum's disease, Sandhoff disease, Schilder's disease, Spinocerebellarataxia, Steele-Richardson-Olszewski disease, peripheral neuropathy,diabetic neuropathy, stroke, depression and Tabes dorsalis.

According to a preferred embodiment of the present invention the peptideis administered to said individual at a dose of 0.1 μg/kg to 20 mg/kgbody weight, preferably 0.5 μg/kg to 10 mg/kg body weight.

Another aspect of the present invention relates to the use of at leastone peptide with neurotrophic and/or neurogenic activity and/or at leastone peptide having an amino acid sequence selected from the groupconsisting of G-D-G-G-L-F-E-K (SEQ ID No. 5), G-L-F-E-K-K-L-W (SEQ IDNo. 6), V-G-D-G (SEQ ID No. 7), G-D-G-G (SEQ ID No. 8), D-G-G-L (SEQ IDNo. 9) and G-G-L-F (SEQ ID No. 10) for the manufacture of a medicamentfor improving learning memory capacities in an individual.

Another aspect of the present invention relates to the use of a moleculeconsisting of a maximum of 50 amino acids with neurotrophic and/orneurogenic activity comprising at least one peptide according to thepresent invention or IPRNEADGMPINV (SEQ ID No. 4) or a fragment thereoffor the manufacture of a medicament for the treatment or enhancement ofmotor deficiencies in an individual.

The present invention is further illustrated by the following figuresand examples, however, without being restricted thereto.

FIG. 1 shows the experimental design.

FIG. 2 shows the proliferation of dentate gyrus progenitor cells in thepresence of peptides 5, 6, 9 and 10 (control).

FIG. 3 shows the analysis of cell proliferation across the 3 layers ofthe dentate gyrus.

FIG. 4 shows newly born cells in oGCL.

FIG. 5 shows the effect of Peptides 5, 9 and 10 on the expression of DCXin BrdU labeled progenitors in the dentate gyrus.

FIG. 6 shows the MAP2 and synaptophysin immunoreactivity in the dentategyrus when Peptide 6 is administered.

FIG. 7 (a) shows the experimental design of example 4; (b) and (c) showthe proliferation of progenitors in four sub-regions of the hippocampus(for anatomical definitions, see “Materials and Methods” section): iGCL(inner granule cell layer, which included the SGZ), oGCL (outer granulecell layer), Mol (molecular layer) and Hil (hilus), revealed thatcompared to control group, CNTF 6c increased the number of BrdU-IR cellsin the iGCL by 45% (p<0.001, Student's t-test), whereas no significantdifferences were observed in either oGCL, Mol or Hil.

FIG. 8 shows the proliferation of immature neurons in the dentate gyrus(a) and the neuronal differentiation of progenitor cells in the dentategyrus (b).

FIG. 9 shows the induction of immediate-early gene expression inresident neurons (a) and neurotrophy and neuroprotection in the dentategyrus.

FIG. 10 shows the enhancement of memory by the administration of CNTF 6aand c.

EXAMPLES Example 1 Dentate Gyrus Neurogenesis and NeurotrophicActivities of Peptides Derived from CNTF in Normal Adult Mice

Materials:

All experiments involving mice were done on 8-10 month old femaleretired breeders of C57BL6 background. A total of 33 mice were dividedinto 10 groups of 3 animals each (except control group which had 6mice). The groups are described in Table 1. Details of CNTF peptides aredescribed in Table 2.

TABLE 1 Group Description Concentration # of mice 1 Control [normal — 6saline] 2 Peptide Mix 0.5 nmol 3 3 Peptide 5-1 0.5 nmol 3 4 Peptide 5-2 5 nmol 3 5 Peptide 6-1 0.5 nmol 3 6 Peptide 6-2  5 nmol 3 7 Peptide 9-10.5 nmol 3 8 Peptide 9-2  5 nmol 3 9 Peptide 10-1 0.5 nmol 3 10 Peptide10-2  5 nmol 3

TABLE 2 CNTF Peptide Position in CNTF MW SEQ ID No. Peptide 5 133-1451384 4 Peptide 6 145-155 1203 1 Peptide 9  91-102 1427 2 Peptide 10 Loop1192 11 (CHQGCGGLFEC)

Experimental Desqin (FIG. 1):

The animals were kept in groups of 3 per cage. The mice were given dailyintraperitoneal injections of four CNTF peptides either separately or ina mixture for 2 weeks as described in Table 1. From day 2, BrdU(Bromodeoxyuridine; 150 mg/Kg) was added to the injections. The animalswere sacrificed 24 hours after the last injection. Briefly, the animalswere perfused transcardially with PBS and their brains taken out anddissected into halves. One hemisphere from each animal was frozen forbiochemical analysis and the other was fixed in 4% paraformaldehyde for48 hours followed by equilibration in 30% sucrose in PBS overnight.These were then processed for immunohistochemistry.

Fixed tissues were cut into 40 μm sections on a freezing slidingmicrotome. One in 5 sections per brain was processed for BrdU stainingand visualized by immunoflorescence. Cell counting was done on thesesections to determine the number of BrdU labeled cells (representingnewly born cells) in the dentate gyrus of the hippocampus. The area ofcounting was limited to the granule cell layer and the subgranular zone(a two-nucleus thick layer adjacent to the granule cell layer). Forcounting purposes, the dentate gyrus was divided into two areas, theouter granule cell layer (OGCL) consisting of one half of the granulecell layer, and the subgranular zone (SGZ) of the inner half (towardsthe hilus) of the granule cell layer plus a two-nucleus thick layeradjacent to the outer border of the hilus. Cell counting was done onconfocal images of the sections according to the optical dissectorprinciple. Volumetric analysis was carried out with the help of ImagePro software.

Results:

The analysis of cell proliferation in the dentate gyrus of mice treatedwith individual peptides is as follows:

1.) Peptides 5, 6 and 9 induced statistically significant increase inthe proliferation of dentate gyrus progenitor cells. Peptides 5 and 6induced more proliferation than peptide 9, whereas Peptide 10 had noeffect on cell proliferation (FIG. 2).

2.) Analysis of cell proliferation across the 3 layers of the dentategyrus showed (FIG. 3):

a.) Peptides 5 and 6 at both concentrations (0.5 and 5 nmoles/animal)increased cell proliferation in the SGZ (subgranular zone) and OGCL(outer portion of the granule cell layer) of the dentate gyrusindicating their effect on both proliferation and migration ofprogenitors.

b.) Peptide 9 at 0.5 nmoles/animal increased cell proliferation in theSGZ with a similar trend at higher concentration of the peptide (5nmoles/animal).

c.) None of the peptides alone had any effect on the number ofprogenitors in the hilus.

3.) There was no evidence of ectopic cell birth in the dentate gyrus asevidenced by lack of selective increase in the percentage of newly borncells in OGCL (FIG. 4).

Example 2 Studies with Cell Based Assay

Cell-Based Assay for Differentiation

βIII-tubulin antigenicity is compromised by fixation, although longerfixation improves attachment to the plates.

Therefore, plates with different coatings for their suitability to beused in differentiation assay using CNTF (0.1, 1, 10, 100 ng/ml) weretested.

Coating Matrices Tested:

-   -   poly D-lysine    -   poly D-lysine/laminin    -   poly L-ornithine/laminin    -   polyethyleneimine    -   collagen I    -   fibronectin

Results:

AHPs did neither divide nor differentiate on collagen I or fibronectin.

The best immunostaining for βIII-tubulin was with 10 min fixation.However, since less cells were lost with 1 hr fixation, thedifferentiation assay with CNTF was performed with 1 hr fixation.

The best differentiation was achieved on polyethyleneimine coated platesusing the ratio of βIII-tubulin immunostaining to DAPI nuclear staining(cell number) as % of control as a relative measure of differentiation.

βIII-tu/DAPI (% of Control) Coating (100 ng/ml CNTF) poly D-lysine 175poly D-lysine/laminin 240 poly L-ornithine/laminin 330 polyethyleneimine 480

CNTF Peptides

6. V-G-D-G-G-L-F-E-K-K-L (SEQ ID No. 1) 2. G-D-G-G-L-F-E-K (SEQ ID No.5) 3. G-L-F-E-K-K-L-W (SEQ ID No. 6) 6A. V-G-D-G (SEQ ID No. 7) 6B.G-D-G-G (SEQ ID No. 3) 6C. D-G-G-L (SEQ ID No. 9) 6D. G-G-L-F (SEQ IDNo. 10)

Example 3

In example 1 it was shown that the CNTF peptides mix and Peptides 5, 6and 9 individually induced statistically significant increase in theproliferation of dentate gyrus progenitor cells. Peptides 5 and 6induced more proliferation than Peptide 9, whereas Peptide 10 had noeffect on cell proliferation. In this example the analysis on cellproliferation and neurotrophy if further evidenced.

Results:

Peptide 6 induced a two fold increase in the differentiation of dentategyrus progenitors into DCX (doublecortin) expressing cells in the 14 daytreatment group. Peptides 5, 9 and 10 did not have any effect on theexpression of DCX in BrdU labeled progenitors in the dentate gyrus (FIG.5).

Peptide 6 caused a statistically significant increase in MAP2 andsynaptophysin immunoreactivity in the dentate gyrus of treated mice asmeasured by mean pixel intensity in the outlined area of interest (FIG.6).

Behavioral tests employing Morris Water maze task-based memoryacquisition, retention and recall paradigms have also been carried out.Two groups of 18 mice each were treated with Peptide 6/Placebocontaining implantable subcutaneous pellets with user specified timedrelease kinetics: 14 days for group 1 and 30 days for group 2. Inparticular, the 30 day group showed significant improvement in memoryacquisition as evaluated by time spent in the target quadrant anddistance covered in the target quadrant in the Morris Water Maze task.

Materials & Methods:

All experiments involving mice were done on 8-10 month old femaleretired breeders of C57BL6 background. A total of 33 mice were dividedinto 10 groups of 3 animals each (except control group which had 6mice). The groups are described in Table 1. Details of CNTF peptides aredescribed in Table 2.

The animals were kept in groups of 3 per cage. The mice were given dailyintraperitoneal injections of four CNTF peptides either separately or ina mixture for 2 weeks as described in table 1. From day 2, BrdU(Bromodeoxyuridine; 150 mg/Kg) was added to the injections. The animalswere sacrificed 24 hours after the last injection (FIG. 1). Briefly, theanimals were perfused transcardially with PBS and their brains taken outand dissected into halves. One hemisphere from each animal was frozenfor biochemical analysis and the other was fixed in 4% paraformaldehydefor 48 hours followed by equilibration in 30% sucrose in PBS overnight.These were then processed for immunohistochemistry.

Fixed tissues were cut into 40 μm sections on a freezing slidingmicrotome. One in 5 sections per brain was processed for BrdU stainingand visualized by immunoflorescence. Cell counting was done on thesesections to determine the number of BrdU labeled cells (representingnewly born cells) in the dentate gyrus of the hippocampus. The area ofcounting was limited to the granule cell layer and the subgranular zone(a two-nucleus thick layer adjacent to the granule cell layer). Forcounting purposes, the dentate gyrus was divided into two areas, theouter granule cell layer (OGCL) consisting of out half of the granulecell layer, and the subventricular zone (SVZ) comprising of the innerhalf (towards the hilus) of the granule cell layer plus a two-nucleusthick layer adjacent to the outer border of the hilus. Cell counting wasdone on confocal images of the sections according to the opticaldissector principle. Volumetric analysis was carried out with the helpof Image Pro software.

Example 4 Enhancement of Adult Hippocampal Neurogenesis and SpatialMemory by a CNTF-Based Tetrapeptide

Materials and Methods

Animals and Housing:

All in vivo studies for characterization of peptides (stereology andbehavioral analysis) were performed on 8-10-month-old female retiredbreeders of C57B16 background. The animals were acclimatized for atleast 3 weeks to exclude occasional pregnant mice from the studies. Micewere group-housed (3 animals per cage) with a 12:12 light:dark cycle andwith free access to food and water. All procedures were conducted inaccordance with approved protocols from our institutional Animal WelfareCommittee.

Construction of CNTF-Based Tetrapeptides:

In the preceding examples an 11-mer peptide based on epitope mapping ofneutralizing antibodies to human CNTF with significant neurogenic andneurotrophic activity in the adult mouse dentate gyrus has beenidentified. Based on this peptide, CNTF 6, a set of four tetrapeptideswith overlapping residues to the sequence of the parent peptide CNTF 6(see Table 3) was further constructed. These peptides, CNTF 6a-d, weresynthesized on a commercial basis by the Pan Biotechnology Facility ofStanford University (Palo Alto, Calif.).

TABLE 3 CNTF Peptide Position in CNTF Peptide 6a 145-148 Peptide 6b146-149 Peptide 6c 147-150 Peptide 6d 148-151

Treatment of Animals with Peptides:

To study neurogenesis, mice received subcutaneous implants of extendedrelease depot pellets containing either CNTF peptides 6a or 6c for 30days of continuous dosing (Innovative Research of America, Sarasota,Fla.). For control groups, the pellets consisted of the carrierbiopolymer only. For implantation, the mice were anesthetized with 2.5%Avertin (0.38 ml for a 25 g animal). Under sterile conditions, thepellets were then subcutaneously implanted along the anterolateralaspect of the right shoulder with a precision trochar (InnovativeResearch of America). The animals were then transferred to the animalcolony after recovery from anesthesia. There were no complicationsassociated with the implantation and treatment. BrdU was given as twodaily i.p. injections (100 mg/kg/dose) for five days starting on day 2of peptide treatment. Neurogenesis was assessed in the dentate gyrus(DG) by counting the number of BrdU-immunoreactive (BrdU-IR),BrdU-DCX-IR and BrdU-NeuN-IR cells in various layers of the DG.Employing principles of unbiased stereology, the optical fractionatormethod was used to estimate cell counts for the DG.

Antibodies:

The following primary antibodies were used forimmunohistochemistry:anti-BrdU (1:400; Accurate) a rat monoclonal raisedagainst BrdU; anti-DCX (1:200; Santa Cruz Biotechnology Inc.), a goatpolyclonal antibody raised against an 18-amino acid peptide representingresidues 384-410 of human doublecortin; anti-NeuN (1:500; Chemicon), amouse monoclonal antibody raised against purified cell nuclei from mousebrain; Anti-c-Fos (Ab-5) (1:500; Calbiochem), a rabbit polyclonalantibody raised against a synthetic peptide corresponding to amino-acids4-17 of human c-Fos; SMI52 (1:1000; Sternberger Monoclonals), a mousemonoclonal antibody specific for the mature neuronal marker MAP2a,b;anti-synaptophysin, SYN (1:200; Chemicon), a mouse monoclonal anti-bodyraised against vesicular fraction of bovine brain. The followingsecondary antibodies were used: Alexa 488-conjugated goat anti-mouse IgGantibody and Alexa 594-conjugated goat anti-rabbit or anti-rat IgGantibody (Molecular Probes); biotinylated anti-rat IgG antibody andCy5-conjugated goat anti-mouse anti-body (Jackson ImmunoResearch).

Tissue Processing:

At the end of treatment, all animals were anesthetized with an overdoseof sodium pentobarbital and transcardially perfused with 0.1 M PBS.After perfusion, the brains were removed from the skull, the lefthemisphere was immediately frozen for future biochemical analysis andthe right hemisphere was fixed in 4% paraformaldehyde in 0.1 M PBS forat least 24 hours at room temperature. Tissues were then stored in 30%sucrose solutions at 4° C. until sectioning. The brains were sectionedsagittaly on a freezing sliding microtome at 40 μm through the entirehippocampus and the sections were stored in glycol anti-freeze solution(Ethylene glycol, glycerol and 0.1 M PBS in 3:3:4 ratio) at −20° C. tillfurther processing.

Immunohistochemistry:

Immunohistochemistry was performed as described elsewhere (Kuhn et al.,J. Neurosci 17 (15) (1997): 5820-5829). Briefly, every 5^(th) brainsection was chosen for quantification of cell number and every 10^(th)section was chosen for staining intensity scanning. Immunohistochemistrywas performed on free floating sections. For BrdU immunohistochemistry,epitope retrieval and staining were performed as previously described(Kuhn et al., J. Neurosci 17 (15) (1997): 5820-5829).

Definitions, Stereology and Confocal Imaging:

Neurogenesis was assessed in the DG by counting the number ofBrdU-immunoreactive (BrdU-IR), BrdU-DCX-IR and BrdU-NeuN-IR cells invarious layers of the DG. The granule cell layer (GCL) was subdividedinto an inner and outer half (iGCL and OGCL). The iGCL consisted of thesubgranular zone (SGZ, defined as a 2-3 nuclei thick layer bordering theGCL) and the inner half of the GCL adjacent to the Hilus (Hil); theouter GCL (OGCL) was defined as the half of the GCL adjacent to theMolecular layer (Mol). A cell in the middle of the GCL was consideredpart of the iGCL and a cell bordering the GCL in the Mol was included inoGCL counts. Mol was defined as the region between the superior limb ofGCL and hippocampal fissure and between the inferior limb of the GCL andthe inferior borders of the DG. Hil included the superficial polymorphiclayer.

All sections were collected using the random uniform sampling scheme.For BrdU-IR cells, counting was performed on every 5^(th) section using40× oil objective of a Nikon 90i fluorescent microscope equipped withNikon C1 three laser confocal system and a Nikon DS U1 digital camera.Employing principles of unbiased stereology, the optical fractionatormethod was used to estimate cell counts for the DG (West et al., AnatRec 231 (1991): 482-497). All layers of the DG described above wereanalyzed separately for cell counting. For each brain, at least 100cells were counted based on coefficient of error determinations.

For BrdU-DCX-, BrdU-NeuN-, and c-Fos-NeuN-IR cells, only GCL (consistingof iGCL and oGCL described above) was counted using 100× oil objectivein every 10^(th) section. To ensure objectivity, z-stacks were collectedfor each double IR cell and analyzed later by generating maximumprojection and 3D constructs. A cell was counted only when it showeddouble IR on 3D reconstructed images.

For MAP2 and Synaptophysin IR, the entire area of GCL was outlined onevery 10^(th) section. Maximum projection images were then generatedbased on confocal z-stacks, and the antibody staining was quantitated bymeasuring mean pixel intensity (MPI) with the help of Image-Pro Plus 5.0software (Media Cybernetics).

All quantitations based on immunohistochemistry were verifiedindependently on coded slides by a second investigator.

Morris Water Maze Task:

For behavioral studies, performance on the Morris Water Maze task wasassessed in three groups of 10 mice each (placebo, CNTF6a and CNTF6c)which received peptide treatment for 30 days. To avoid daily stress dueto injections, all animals undergoing behavioral studies receivedsubcutaneous implants of CNTF 6a, CNTF 6c or placebo pellets asdescribed above.

All animals for behavioral testing were coded such that theexperimentator was blind to the assignment of the animals to specifictreatment groups. The Morris Water Maze procedure was performed using a110 cm diameter circular tank. Before training, the mice were handledgently for 2-3 min/day during 3 days to minimize non-specific stress.Acquisition was started with the submerged (invisible) escape platformin the North-East quadrant and each animal was given 60 sec to find thesubmerged escape platform. If the mouse did not find the platform in 60sec, it was guided to it. Five such acquisition trials were given oneach day, for four consecutive days. A test for retention, or probetrial, was given 24 hours later. During the probe trial the mouse wasallowed to swim in the tank without the escape platform for 60 seconds.This was followed by second and third probe trials 15 and 30 days fromthe first probe trial. Each probe trial was immediately followed by a“retraining session” consisting of 5 trials/animal to consolidatelearned behavior.

The measures of learning were the time and distance swum to reach theescape platform. For retention during the probe trial, the tank wasdivided into four imaginary quadrants and a small zone where the escapeplatform had been (virtual platform). The measures of retention were thepercent of time spent and the percent of distance swum in each quadrant,and the number of entries into the platform zone.

Mouse behavior in the Morris Water Maze was monitored by a SamsungDigital Camera (SDC 4304) mounted to the ceiling and tracked and timedby a SMART (Pan Lab/San Diego Instruments) version 2.0.14 software.

Statistics:

Data are represented as mean±SEM. For analysis involving multiplegroups, ANOVA with post hoc Tukey's test was used. For analysis of datawith skewed distributions, the nonparametric Mann-Whitney U-test wasused. For all other comparisons (including inter-group comparisons),Student's t-test was used. Differences with p<0.05 were consideredsignificant.

Results:

Proliferation of Neural Progenitor Cells with Synthetic Peptides:

The four CNTF tetrapeptides were initially screened in a behavioralparadigm employing the Morris Water Maze. Two CNTF tetrapeptides, CNTF6a and CNTF 6c, were chosen for detailed stereological and behavioralanalysis.

Fifteen mice were divided into 3 groups including placebo, CNTF 6a andCNTF 6c. Mice received subcutaneous implants of 30-day extended releasepellets containing either CNTF 6a or CNTF 6c (50nmol/peptide/animal/day, n=5/group) or placebo (n=5). Dividing cellswere labeled with BrdU given i.p. for five days, twice a day (100mg/kg/animals/dose; FIG. 7 a). Compared to the placebo group, CNTF 6cincreased BrdU-immunoreactive (BrdU-IR) cell counts in the GCL by 31%(p<0.05, Student's t-test). CNTF 6a had not significant effect on cellproliferation in the GCL (FIG. 7 b, c and Table 2).

Further examination of the proliferation in four sub-regions of thehippocampus (for anatomical definitions, see “Materials and Methods”section): iGCL (inner granule cell layer, which included the SGZ), oGCL(outer granule cell layer, Mol (molecular layer) and Hil (hilus),revealed that compared to control group, CNTF 6c increased the number ofBrdU-IR cells in the iGCL by 45% (p<0.001, Student's t-test), whereas nosignificant differences were observed in either OGCL, Mol or Hil (FIGS.7 b and c, Table 4). CNTF 6a had no effect on BrdU-IR cell numbers ineither of the four sub-regions of the DG. Together, these data suggestthat both CNTF 6c increased BrdU-IR cells in the DG and this increasewas mainly confided to the iGCL, the neurogenic niche of thehippocampus.

TABLE 4 Stereological counts (±SEM) of BrdU-IR cells in varioussubregions of the hippocampus in 30-day treated mice (n = 5/group) GCLiGCL oGCL Mol Hil Control 427 ± 38  334 ± 28  93 ± 19 526 ± 77 108 ± 17CNTF 6a 493 ± 28  382 ± 15*  110 ± 13  538 ± 99 126 ± 8  CNTF 6c 560 ±24* 486 ± 31** 74 ± 10 487 ± 28 121 ± 16 *p < 0.05, **p < 0.01,Student's t-test

Proliferation of Immature Neurons in the Dentate Gyrus:

Doublecortin (DCX), an immature neuronal marker, is used to quantitateearly neuronal fate determination in DG progenitors. The number ofDCX-IR cells in the GCL (iGCL+OGCL) was quantitated at the time ofperfusion, a snapshot-quantitation of immature neurons in response to30-day treatment with CNTF tetrapeptides (FIG. 8 a). Stereologicalanalysis revealed that compared to the placebo, CNTF 6c treatmentincreased DCX-IR cells in the GCL by almost 2 folds (˜91%, increase,p<0.001, Student's t-test), whereas CNTF 6a treatment did not show anysignificant difference (FIG. 8 a and Table 5). These data suggest thatat the time of perfusion, there were more immature neurons in the GCL ofCNTF 6c treated animals. Whether this also reflects early neuronaldifferentiation of dividing progenitors cannot be determined by ourstudy.

TABLE 5 Stereological counts (±SEM) of cells expressing various neuronalmaturity and/or activity markers in the granule cell layer of thedentate gyrus in 30-day treated mice DCX NeuN-BrdU/BrdU c-fos-NeuNControl 306 ± 72 24 ± 2 168 ± 17 CNTF 6a 360 ± 33 19 ± 2 214 ± 27 CNTF6c  656 ± 43**  39 ± 2**  247 ± 23* (n = 5/group) *p < 0.05, **p < 0.01,Student's t-test

Neuronal Differentiation of Progenitor Cells in the Dentate Gyrus:

Net neurogenesis in the DG is determined by the number of progenitorswhich survive as mature neurons, as more than half of the progenitorseither die as stem cells or as immature precursors (eg. DCX-IR cells).In order to determine whether CNTF 6c induced differentiation of DGprogenitors into mature neurons, the number of BrdU-IR cells expressingthe mature neuronal marker NeuN in the GCL of the DG was counted. A 62%increase in BrdU-NeuN-IR cells in CNTF 6c treated animals was found whencompared with the placebo group, whereas CNTF 6a treatment had no effect(p<0.01, Stundent's t-test; FIG. 8 b and Table 5).

Induction of Immediate-Early Gene Expression in Resident Neurons:

For neurogenesis to have physiological significance, newly born neuronsneed to be functionally integrated into the hippocampal circuitry.Neuronal activity, an indication of functional integration, can beindirectly quantitated by studying changes in the expression ofimmediate-early genes like c-fos and zif. Towards that aim, weinvestigated whether CNTF 6c induced an increase in c-fos proteinexpression, providing a biological substrate for neuronal firing, andultimately spatial encoding. Stereological counts of c-fos expressingmature DG neurons without behavioral stimulation, i.e. at basal levelsreflecting activity in the cage (FIG. 9 a and Table 5) were compared. Itwas found a ˜47% increase in the number of mature neurons (NeuN-IR)co-expressing c-fos in the GCL in CNTF 6c treated mice (p<0.05,Student's t-test). There was also evidence of increased neuronalactivity in newly born mature neurons as some BrdU-NeuN-IR cells in theGCL also co-expressed zif (FIG. 9 a).

Neurotrophy and Neuroprotection in the Dentate Gyrus:

Microenvironment within the brain undergoes significant changes in bothaging and disease. The rate of neurogenesis and synaptogenesis in thebrain indirectly reflect its microenvironment. In order to study whetherCNTF 6c-induced enhancement of DG neurogenesis was also accompanied bychanges in local neurotrophy, the expression of MAP2 and synaptophysin,indicators of dendritic arborization and synaptic activity respectively,in the GCL of treated animals was measured. An increase in bothindicators of neurotrophy (31% and 26% respectively, p<0.01, Student'st-test) as measured by mean-pixel intensity was found (FIG. 9 b).

Enhancement of Memory:

Increased neuronal differentiation of DG progenitors, enhanced neuronalfiring, upregulated synaptogenesis and neurotrophy are all keybiological substrates of memory processing within the DG. Therefore, itwas evaluated whether CNTF 6c treatment also had an effect on thecognitive function of treated animals. Since normal adult mice were usedas experimental animals, it was crucial not to miss any effect on memoryacquisition and learning that the 30-day peptide treatment might havehad. Therefore, a partial training paradigm was used to evaluatelearning and memory in the Morris Water Maze. Treated mice were trainedon the Morris Water Maze for a total of 20 sessions spanning 4 daysafter which they were subjected to the first probe trial (P1). Twoadditional probe trials (P2 and P3) were administered 15 and 30 daysafter P1. Each probe trial was immediately followed by 4 retrainingsessions to allow memory consolidation (FIG. 10 a). Learning wasevaluated in terms of latency and distance traveled to reach theinvisible escape platform. Retention was measured on probe trials by thepercent of time and travel distance in the target quadrant, and thenumber of crossings of the virtual platform.

Animals in all tree groups learned well as evident by declining swimlatencies to reach the submerged platform (FIG. 10 a). However, therewas no effect of either CNTF 6a or CNTF 6c treatment on learning in thespatial reference memory task (two way ANOVA, p=0.667).

Analysis of retention on the three probe trials showed no effect of thetreatment on P1, whereas P2 and P3 showed significant differences inboth measures of retention in CNTF 6c treated mice. Analysis of timespent in the target quadrant across three probe trials indicated thatwhereas all animals spent equal amount of time on P1, both placebo andCNTF 6a treated animals reduced this time during subsequent P2 and P3.CNTF 6c-treated animals however, spent the same percent amount of timein the target quadrant during the three probe trials, indicating betterpreservation of the memory trace in these mice (FIG. 10 b). Analysis ofthe percent distance traveled within the target quadrant also presenteda similar picture for CNTF 6c across the three probe trials (FIG. 10 c).

Together, these data indicate better consolidation of learned behaviordue to CNTF 6c treatment.

1.-17. (canceled)
 18. A neurotrophic peptide comprising an amino acidsequence consisting of VGDGGLFEKKL (SEQ ID No. 1), EDQQVHFTPTEG (SEQ IDNo. 2), or IPENEADGMPATV (SEQ ID No. 3).
 19. A pharmaceuticalcomposition comprising at least one neurotrophic peptide further definedas a peptide of claim 18 or comprising a fragment comprising 4 to 10amino acids thereof in a pharmaceutically acceptable excipient and/orcarrier.
 20. The composition of claim 19, further defined as comprisingat least one peptide comprising an amino acid sequence of GDGGLFEK (SEQID No. 5), GLFEKKLW (SEQ ID No. 6), VGDG (SEQ ID No. 7), GDGG (SEQ IDNo. 8), DGGL (SEQ ID No. 9), and/or GGLF (SEQ ID No. 10).
 21. Thecomposition of claim 19, further defined as comprising apharmaceutically acceptable excipient and/or carrier.
 22. Thecomposition of claim 19, wherein the composition is suitable forintravenous, intramuscular, spinal, epidural, transdermal, subcutaneous,parenteral, intranasal, mucosal, oral, enteral, and/or rectaladministration.
 23. The composition of claim 19, wherein the compositionis further defined as a slow release formulation.
 24. The composition ofclaim 19, wherein the peptide is comprised in the composition in anamount between 0.1 μg/g to 100 mg/g.
 25. The composition of claim 24,wherein the peptide is comprised in the composition in an amount between1 μg to 80 mg/g.
 26. A method of treating a subject comprising:obtaining a pharmaceutical composition of claim 19; and providing thecomposition to a subject; wherein a neurodegenerative disease is treatedand/or prevented in the subject, learning-memory capacity is improved inthe subject, and/or motor deficiencies are treated or enhanced in thesubject.
 27. The method of claim 26, further defined as a method oftreating and/or preventing a neurodegenerative disease in the subject.28. The method of claim 27, wherein the neurodegenerative disease isAlexander disease, Alper's disease, Alzheimer disease, Amyotrophiclateral sclerosis, Ataxia telangiectasia, Canavan disease, Cockaynesyndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease,epilepsy, Huntington disease, Kennedy's disease, Krabbe disease, Lewybody dementia, Machado-Joseph disease (Spinocerebellar ataxia type 3),Multiple sclerosis, Multiple System Atrophy, Parkinson disease,Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral sclerosis,Refsum's disease, Sandhoff disease, Schilder's disease, Spinocerebellarataxia, Steele-Richardson-Olszewski disease, peripheral neuropathy,diabetic neuropathy, stroke, depression, or Tabes dorsalis.
 29. Themethod of claim 26, further defined as a method of improvinglearning-memory capacities in the subject.
 30. The method of claim 26,further defined as a method of treatment or enhancement of motordeficiencies in the subject.
 31. The method of claim 26, wherein thecomposition further comprises at least one additional pharmaceuticallyactive component.
 32. The method of claim 26, wherein the composition isprovided intravenously, intramuscularly, spinally, epidurally,intranasally, mucosally, transdermally, subcutaneously, parenterally,orally, enterally, or rectally.
 33. The method of claim 26, wherein thecomposition a slow release formulation.
 34. The method of claim 26,wherein the composition comprises the peptide in an amount between 0.1μg/g to 100 mg/g.
 35. The method of claim 34, wherein the compositioncomprises the peptide in an amount between preferably 1 μg to 80 mg/g.36. The method of claim 35, wherein the peptide is administered to thesubject at a dose of 0.1 μg/kg to 20 mg/kg body weight.
 37. The methodof claim 36, wherein the peptide is administered to the subject at adose of 0.5 μg/kg to 10 mg/kg body weight.
 38. The method of claim 26,wherein the subject is a human.